Ion channels are molecules that form water-filled pores in membranes through which ions flow. They have been critically implicated in a wide variety of biological processes. One class of ion channel opens and closes in response to changes in transmembrane voltage to regulate the flow of ions. The structure of these channels, the conformational changes associated with the transitions from open to closed, and the nature of the voltage-sensing domain of the channel protein are all poorly understood despite much speculation and theory. This proposal is aimed at extending our understanding of the structure and function of one class of voltage-gated channel, the VDAC channel found in the outer mitochondrial membrane, as a model of the general phenomena of voltage-gating of ion flow. The unique and powerful system that has been established exploits the relative simplicity of the VDAC channel and the power of molecular genetic analysis as is possible in yeast. This system has allowed the existing manipulation of the VDAC protein in a variety of ways in order to develop a detailed model of the structure of the open channel. To extend these observations, the collection of single and multiple amino acid substitution mutants will be examined to elucidate the structure of the closed channel and identify regions of the protein which form the voltage sensor. Molecular genetic tools will also allow manipulation of the VDAC protein in new ways to test predictions concerning the structure and function of this channel and to analyze, in detail, the sequence requirements of the voltage sensor. Antibody probes will be developed to independently test and constrain models of VDAC structure. Finally, the genes coding for VDAC from Drosophila and rat will be isolated and characterized. Comparisons of these amino acid sequences should point out conserved structural features responsible for VDAC's evolutionarily conserved channel properties. This study should result, then, in a detailed understanding of the nature of the voltage sensor and conformational transitions of one voltage-gated channel, complimenting and expanding our knowledge of this important class of proteins.